Laulimalide (1) and isolaulimalide (2), also known as fijianolides, were originally isolated as cytotoxic agents from the Indonesian sponge Hyatella sp. (E. Quinoa et al., xe2x80x9cFijianolides, polyketide heterocycles from a marine sponge,xe2x80x9d J. Org. Chem., 1988, 53, 3642; D. G. Corley et al., xe2x80x9cLaulimalides. New potent cytotoxic macrolides from a marine sponge and a nudibranch predator,xe2x80x9d J. Org. Chem. 1988, 53, 3644-3646), and later found along with neolaulimalide (3) in the Okinawan sponge Fasciospongia rimosa (Jefford et al., xe2x80x9cStructures and absolute configurations of the marine toxins, latrunculin A and laulimalide,xe2x80x9d 1996, Tetrahedron Letts. 37: 159-162; Higa et al., xe2x80x9cThree new cytotoxic macrolides from a marine sponge,xe2x80x9d PCT publication No. WO 97/10242). The absolute structure of natural (xe2x88x92)-laulimalide has been determined by X-ray crystallography. 
A number of total syntheses of laulimalide have been reported. See Ghosh et al., 2001, xe2x80x9cTotal synthesis of microtubule-stabilizing agent (xe2x88x92)-laulimalide,xe2x80x9d J. Org. Chem. 66: 8973-8982; Paterson et al., 2001, xe2x80x9cTotal synthesis of microtubule-stabilizing agent (xe2x88x92)-laulimalide,xe2x80x9d Org. Letts.: 3149-3152; Enev et al., 2001, xe2x80x9cMacrocyclization via allyl transfer: total synthesis of laulimalide,xe2x80x9d J. Am. Chem. Soc. 123: 10764-10765; Ghosh et al., 2000, xe2x80x9cTotal synthesis of(xe2x88x92)-laulimalide,xe2x80x9d J. Am. Chem. Soc. 122: 11027-11028.
A mechanism-based screening program aimed at isolating novel microtubule-directed anticancer agents revealed both laulimalide and isolaulimalide to be potent stabilizers of microtubules, similar to paclitaxel. See S. Mooberry et al., 1999, Cancer Res., 59, 653-660; Mooberry and Davidson, xe2x80x9cLaulimalide compounds as microtubule stabilizing agents,xe2x80x9d PCT Publication No. WO 01/54689, incorporated herein by reference.
Because of the problems associated with the use of paclitaxel such as low solubility and resistance, there is an increasing need for alternative anti-cancer compounds. The present invention provides such compounds.
The present invention provides laulimalide compounds, intermediates thereto and methods for their preparation, and methods for their use in the treatment of diseases characterized by cellular hyperproliferation.
The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds that are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term xe2x80x9cadministeringxe2x80x9d shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in xe2x80x9cDesign of Prodrugsxe2x80x9d, ed. H. Bundgaard, Elsevier, 1985.
Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
The term xe2x80x9calkylxe2x80x9d refers to optionally substituted straight, branched or cyclic hydrocarbons comprising no unsaturation. xe2x80x9cAlkenylxe2x80x9d refers to an optionally substituted straight, branched, or cyclic chain hydrocarbon with at least one carbon-carbon double bond. xe2x80x9cAlkynylxe2x80x9d refers to an optionally substituted straight, branched, or cyclic hydrocarbon with at least one carbon-carbon triple bound. Substituted alkyl, substituted alkenyl, or substituted alkynyl refer to the respective alkyl, alkenyl or alkynyl group substituted by one or more substituents. Illustrative examples of substituents include but are not limited to alkyl, alkenyl, alkynyl, aryl, halo; trifluoromethyl; trifluoromethoxy; hydroxy; alkoxy; cycloalkoxy; heterocyclooxy; oxo (xe2x95x90O); alkanoyl (xe2x80x94C(xe2x95x90O)-alkyl); aryloxy; alkanoyloxy; amino; alkylamino; arylamino; aralkylamino; cycloalkylamino; heterocycloamino; disubstituted amines in which the two amino substituents are selected from alkyl, aryl, or aralkyl; alkanoylamino; aroylamino; aralkanoylamino; substituted alkanoylamino; substituted arylamino; substituted aralkanoylamino; thiol; alkylthio; arylthio; aralkylthio; cycloalkylthio; heterocyclothio; alkylthiono; arylthiono; aralkylthiono; alkylsulfonyl; arylsulfonyl; aralkylsulfonyl; sulfonamido (e.g., SO2NH2); substituted sulfonamido; nitro; cyano; carboxy; carbamyl (e.g., CONH2); substituted carbamyl (e.g., xe2x80x94C(xe2x95x90O)NRxe2x80x2Rxe2x80x3 where Rxe2x80x2 and Rxe2x80x3 are each independently hydrogen, alkyl, aryl, aralkyl and the like); alkoxycarbonyl, aryl, guanidino, and heterocyclo such as indoyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Where applicable, the substituent may be further substituted such as with halogen, alkyl, alkoxy, aryl, or aralkyl and the like.
The term xe2x80x9carylxe2x80x9d refers to an optionally substituted aromatic ring having 6 to 12 carbon atoms and may include one or more heteroatoms such as N, S and O. Illustrative examples of aryl include but are not limited to biphenyl, furyl, imidazolyl, indolyl, isoquinolyl, naphthyl, oxazolyl, phenyl, pyridyl, pyrryl, quinolyl, quinoxalyl, tetrazoyl, thiazoyl, thienyl and the like. Substituted aryl refers to an aryl group substituted by, for example, one to four substituents such as substituted and unsubstituted alkyl, alkenyl, alkynyl, and aryl; halo; trifluoromethoxy; trifluoromethyl; hydroxy; alkoxy; cycloalkyloxy; heterocyclooxy; alkanoyl; alkanoyloxy; amino; alkylamino; aralkylamino; cycloalkylamino; heterocycloamino; dialkylamino; alkanoylamino; thio; alkylthio; cycloalkylthio; heterocyclothio; ureido; nitro; cyano; carboxy; carboxyalkyl; carbamyl; alkoxycarbonyl; alkylthiono; arylthiono; alkylsulfonyl; sulfonamido; aryloxy; and the like. The substituent may be further substituted, for example, by halo, hydroxy; alkyl, alkoxy; aryl, substituted aryl, substituted alkyl, substituted aralkyl, and the like.
The terms xe2x80x9chalogen,xe2x80x9d xe2x80x9chaloxe2x80x9d, or xe2x80x9chalidexe2x80x9d refer to fluorine, chlorine, bromine and iodine.
The term xe2x80x9csubjectxe2x80x9d as used herein, refers to an animal, preferably a mammal, or, most preferably, a human, that is the object of treatment, observation or experiment.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
As used herein, the term xe2x80x9ccompositionxe2x80x9d is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
In one aspect, the present invention provides compounds of the formula 
wherein:
X is O or NR, wherein R is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, alkylaryl, alkenylaryl, or alkynylaryl;
Z is O, CH2, or a bond;
R1 is hydrogen, hydroxyl, C1-C10 alkoxy, aryloxy, or alkylaryloxy;
R2 is hydrogen, hydroxyl, C1-C10 alkoxy, aryloxy, or alkylaryloxy; and
R3 is cyclohexyl, 3-cyclohexenyl, phenyl, or a group of the formula 
In one embodiment of the invention, the laulimalide compounds of formula (I) are provided 
wherein:
X is O or NR, wherein R is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, arylalkyl, arylalkenyl, or arylalkynyl;
R1 is H, OH, C1-C10 alkoxy, aryloxy, or alkylaryloxy;
R2 is H, OH, C1-C10 alkoxy, aryloxy, or alkylaryloxy; and
R3 is cyclohexyl, 3-cyclohexenyl, phenyl, or a group of the formula 
In another embodiment of the invention, compounds of formula (I) are provided wherein
Xxe2x95x90O or NR, wherein R is H, C1-C5 alkyl, phenyl, or benzyl;
R1 is OH, or C1-C5 alkoxy;
R2 is H, OH, or C1-C5 alkoxy; and
R3 is cyclohexyl, 3-cyclohexenyl, phenyl, or a group of the formula 
In another embodiment of the invention, compounds of formula (I) are provided wherein
Xxe2x95x90O or NH;
R1 is OH, or C1-C5 alkoxy;
R2 is H, OH, or C1-C5 alkoxy; and
R3 is a group of the formula 
In another embodiment of the invention, the compounds of formula (I) having the formulas 
are provided.
In another embodiment of the invention, compounds of formulas (II) are provided 
wherein:
X is O or NR, wherein R is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, arylalkyl, arylalkenyl, or arylalkynyl;
R1 is H, OH, C1-C10 alkoxy, aryloxy, or alkylaryloxy;
R2 is H, OH, C1-C10 alkoxy, aryloxy, or alkylaryloxy; and
R3 is cyclohexyl, 3-cyclohexenyl, phenyl, or a group of the formula 
In another embodiment of the invention, compounds of formula (II) are provided wherein
Xxe2x95x90O or NR, wherein R is H, C1-C5 alkyl, phenyl, or benzyl;
R1 is OH, or C1-C5 alkoxy;
R2 is H, OH, or C1-C5 alkoxy; and
R3 is cyclohexyl, 3-cyclohexenyl, phenyl, or a group of the formula 
In another embodiment of the invention, compounds of formula (II) are provided wherein
Xxe2x95x90O or NH;
R1 is OH, or C1-C5 alkoxy;
R2 is H, OH, or C1-C5 alkoxy; and
R3 is a group of the formula 
In another embodiment of the invention, the compounds of formula (II) having the formulas 
are provided.
In another embodiment of the invention, compounds of formulas (III) are provided 
wherein:
X is O or NR, wherein R is H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, arylalkyl, arylalkenyl, or arylalkynyl;
R2 is H, C1-C10 alkoxy, aryloxy, or alkylaryloxy; and
R3 is cyclohexyl, 3-cyclohexenyl, phenyl, or a group of the formula 
In another embodiment of the invention, compounds of formula (III) are provided wherein
Xxe2x95x90O or NR, wherein R is H, C1-C5 alkyl, phenyl, or benzyl;
R2 is H or C1-C5 alkoxy; and
R3 is cyclohexyl, 3-cyclohexenyl, phenyl, or a group of the formula 
In another embodiment of the invention, compounds of formula (III) are provided wherein
Xxe2x95x90O or NH;
R2 is H or C1-C5 alkoxy; and
R3 is a group of the formula 
In another embodiment of the invention, the compounds of formula (III) having the formulas 
are provided.
The compounds of the present invention possess paclitaxel-like microtubule stabilizing activity and are potent inhibitors of cell proliferation. Based on initial studies on paclitaxel-resistant cell line SKVLB-1, these compounds are expected to be active against paclitaxel-resistant tumors since they appear to be poor substrates for P-glycoprotein mediated transport. In addition to being anti-cancer agents, the compounds of the present invention may be used to treat other diseases that are characterized by cellular hyperproliferation such as many inflammatory disorders, for example psoriasis, eczema, dermatitis, multiple sclerosis, and rheumatoid arthritis, and restenosis. The compounds of the invention have further utility to kill cells, ameliorate the detrimental effects of cell growth, and generally to substitute for any other cytotoxic agent in any application thereof.
The compounds of the present invention are expected to be more resistant to degradation than is laulimalide. Isolaulimalide (which is a less potent stabilizer of microtubule polymerization than laulimalide) is a degradation product of laulimalide, formed by attack of the 20-OH on the reactive 16,17-epoxide. Neolaulimalide may also be a degradation product resulting from translactonization from O19 to O20. Alterations to the laulimalide structure that would block such degradative processes are expected to increase the effectiveness of laulimalide as an antimitotic agent.
Thus, in one embodiment of the invention, the 16,17-epoxide is replaced with an alkene to form 16,17-desoxylaulimalide. It is likely that this change would increase the therapeutic index as well as increase the stability of laulimalide, based on similar findings with epothilone B and its desoxy-analog, epothilone D.
In another embodiment of the invention, the 16,17-epoxide is replaced with a cyclopropane in order to stabilize laulimalide against rearrangement to isolaulimalide.
In another embodiment of the invention, the reactive 20-OH group is either removed or blocked as a 20-O-alkyl ether derivative. Removal of the nucleophilic 20-OH group will prevent rearrangement of laulimalide to isolaulimalide.
In another embodiment of the invention, the lactone group is replaced with a lactam to give laulimalide lactams. Laulimalide lactams are expected to be resistant towards formation of neolaulimalides.
In another embodiment of the invention, the modifications described above are combined to produce highly stabilized laulimalide compounds. Thus, for example, the lactam analogs of 16,17-desoxylaulimalide, cyclopropyl laulimalides, 20-deoxylaulimalides, and 20-O-alkyl ether laulimalides will be stabilized against degradation to both isolaulimalides and neolaulimalides.
In another aspect of the invention, methods for the conversion of laulimalide into more stable derivatives are provided. Laulimalide itself is available either through total synthesis as described above or through isolation from natural sources, as described in Quinoa et al., xe2x80x9cFijianolides, polyketide heterocycles from a marine sponge,xe2x80x9d J. Org. Chem. 1988, 53: 3642-3; Corley et al., xe2x80x9cLaulimalides. New potent cytotoxic macrolides from a marine sponge and a nudibranch predator,xe2x80x9d J. Org. Chem. 1988, 53: 3644-6; and Mooberry and Davidson, xe2x80x9cLaulimalide compounds as microtubule stabilizing agents,xe2x80x9d PCT publication WO 01/54689; each of which is incorporated herein by reference in its entirety.
In one embodiment, laulimalide is deoxygenated using titanocene dichloride (TiCp2Cl2) and magnesium to provide 16,17-desoxylaulimalide. Similar reagents useful for this transformation include that of Sharpless, tungsten hexachloride (WCl6) and n-butyllithium, and that of Yadav et al., titanocene dichloride with zinc and zinc chloride. In one form of this embodiment, laulimalide is directly treated with the deoxygenation reagent to provide 16,17-desoxylaulimalide. In a preferred form of the embodiment, laulimalide (1) is first reacted with a silylating agent, for example tert-butyldimethylsilyl chloride or triflate, in the presence of a mild base such as imidazole or 2,6-lutidine, so as to protect the 15- and 20-hydroxyl groups as their silyl ethers (4) as illustrated in Scheme 1. Compound (4) is then reacted with the deoxygenating reagent to produce the protected compound (5). Subsequent desilylation with HF would provide 16,17-desoxylaulimalide (6). 
In another embodiment, methods for conversion of 16,17-desoxylaulimalide into the cyclopropane derivative 16,17-desoxy-16,17-methylenelaulimalide (7) are provided. Cyclopropanation is affected using the Simmons-Smith procedure as illustrated in Scheme 2. This reagent (CH2I2 with a Zn/Cu couple) and later improvements thereof, for example Et2Zn and CH2I2, rely upon the directing effect of an adjacent alcohol to deliver a CH2 equivalent to the olefin of an allylic alcohol. There are two such allylic alcohols present in 16,17-deoxylaulimalide (positions 15 and 20), leading to possible formation of two monocyclopropanes and a bis(cyclopropane), yet the 16,17-alkene is known to be more reactive. 
In other embodiments of the present invention, methods for preparation of laulimalides modified at the 20-position are provided. In general, these methods involve initial conversion of a laulimalide compound into a 15,20-diester derivative (8) as illustrated in Scheme 3. 
In one embodiment, deoxygenation is performed by treatment of a 20-O-ester with a metal catalyst and a hydride source as illustrated in Scheme 4. Subsequent deprotection of the 15-OH provides 20-deoxylaulimalide (10). Methods for deoxygenating the 20-OH include but are not limited to reaction of a 20-O-cinnamate ester (8, R4xe2x95x90PhCHxe2x95x90CHxe2x80x94) with triethylsilane and (Ph3P)3RhCl, and treatment of the 20-O-acetate ester (8, R4xe2x95x90Me) with formic acid and a palladium catalyst. In another embodiment, the epoxide of the 20-deoxylaulimalide is deoxygenated according to the methods described above to provide 20-deoxy-16,17-disoxylaulimalide (12) after deprotection. 
In another embodiment of the invention, methods for the preparation of 20-O-alkyl ethers (13) of laulimalide are provided. A laulimalide 15,20-dicarbonate (8, R4xe2x95x90OR5) is treated with a palladium catalyst, for example tetrakis(triphenylphosphine)palladium, to effect nucleophilic replacement of the 20-oxygen by the alkoxy group OR5 as illustrated in Scheme 5. The alkyl group R5 of the ether can be varied by changing the carbonate. In a preferred embodiment, R5 is methyl. In another embodiment of the invention, the 16,17-epoxide is deoxygenated, and the resulting 15-carbonate either cleaved to yield (14) or resubmitted to the palladium reaction to generate a 15,20-bisether (15). In another embodiment, compound (14) is converted into a new 15-O-carbonate and then subjected to the palladium reaction so as to provide a 15,20-bisether (15) wherein the R5 groups at 15 and 20 are different. 
In another aspect of the invention, laulimalide analogs are made by de novo synthesis. Scheme 6 illustrates the disconnection of laulimalide into two fragments, A and B, which are used to prepare laulimalide analogs by total synthesis according to the methods described in Paterson et al., xe2x80x9cTotal synthesis of the microtubule-stabilizing agent (xe2x88x92)-laulimalide,xe2x80x9d Org. Letts/2001, 3: 3149-3152. 
In one embodiment of the invention, 20-deoxylaulimalides are prepared by use of the A fragment wherein R2xe2x95x90H. Scheme 7 illustrates a method for preparation of this A fragment. 
Aldol condensation of compound (16), prepared as described in Paterson et al., with aldehyde R3CHO followed by dehydration of the aldol adduct using methanesulfonyl chloride and triethylamine introduces the R3 group. For example, condensation with benzaldehyde will yield the fragment wherein R3 is a phenyl group, condensation with cyclohexanecarboxaldehyde will yield the fragment wherein R3 is a cyclohexyl group, condensation with 3-pyridinecarboxaldehyde will yield the fragment wherein R3 is a 3-pyridyl group, and condensation with thiazole-4-carboxaldehyde will yield the fragment wherein R3 is a 4-thiazolyl group. Substituted aldehydes are used similarly to provide fragments where R3 is e.g., a substituted phenyl, cyclohexyl, cyclohexenyl, pyridyl, or thiazolyl group. The resulting ketone is selectively reduced using zinc borohydride, and the alcohol so produced is acetylated using acetic anhydride and pyridine to provide compound (17). The allylic acetate is reduced using a palladium catalyst such as tetrakis-(triphenylphosphine)palladium and a hydride donor, such as triethylsilane, formic acid, or the like. Finally, the terminal aldehyde is installed by removal of the tert-butyldiphenylsilyl protecting group and oxidation of the resulting alcohol, for example using the Dess-martin periodinane, Swern oxidation (oxalyl chloride, DMSO, triethylamine), or the like, to produce fragment A wherein R2xe2x95x90H.
In one embodiment of the invention, 20-O-alkyl ether laulimalides are prepared by use of the A fragment wherein R2xe2x95x90OR5. Scheme 8 illustrates a method for preparation of this A fragment. 
The R3 group is introduced, and the ketone reduced as discussed above. The alcohol is reacted with an alkyl chloroformate and pyridine to produce the carbonate (17), which is treated with a palladium catalyst such as tetrakis(triphenylphosphine)palladium to produce the ether. Elaboration of the terminal aldehyde as discussed above provides the A fragment wherein R2xe2x95x90OR5.
Scheme 9 illustrates the use of the above-described fragments in the preparation of compounds of the invention. 
Aldol condensation of the A and B fragments using the boron enolate is followed by conversion of the methyl ester to the acid via a three-step sequence of reduction to the alcohol (diisobutylaluminum hydride at xe2x88x9278xc2x0 C.), oxidation of the alcohol to an aldehyde (Dess-Martin periodinane), and oxidation of the aldehyde to the carboxylic acid (NaClO2 in the presence of disodium phosphate, 2-methyl-2-butene, and tert-butanol). The PMB protecting group is removed by treatment with dichlorodicyanoquinone (DDQ) in buffered dichloromethane. The lactone is formed by Mitsunobu inversion using diethyl azodicarboxylate (DEADCAT) and triphenylphosphine. The 13-ketone is then converted into the 13-methylidene group using the Takai reagent (zinc, lead iodide, diiodomethane, and titanium tetrachloride in THF).
In another embodiment of the invention, laulimalide lactams are prepared through total synthesis according to the retrosynthetic analysis shown in Scheme 10. 
The laulimalide lactam is prepared from two main fragments of the molecule, an azide-containing xe2x80x9cfragment Cxe2x80x9d and a ketone-containing xe2x80x9cfragment D.xe2x80x9d In one embodiment of the invention, fragment C wherein R2 is tert-butyldimethylsilyloxy (OTBS) is prepared starting from compound (16) described above as illustrated in Scheme 11. 
Aldol condensation of (16) with an aldehyde via the boron enolate adds the R3 group as described above. For example, condensation with benzaldehyde will yield the fragment wherein R3 is a phenyl group, condensation with cyclohexanecarboxaldehyde will yield the fragment wherein R3 is a cyclohexyl group, condensation with 3-pyridinecarboxaldehyde will yield the fragment wherein R3 is a 3-pyridyl group, and condensation with thiazole-4-carboxaldehyde will yield the fragment wherein R3 is a 4-thiazolyl group. Substituted aldehydes are used similarly to provide fragments where R3 is e.g., a substituted phenyl, cyclohexyl, cyclohexenyl, pyridyl, or thiazolyl group. The intermediate aldol adduct is dehydrated using methanesulfonyl chloride and an amine base. The ketone group is reduced diastereoselectively using zinc borohydride at xe2x88x9240xc2x0 C., and the resulting alcohol is protected as its tert-butyldimethylsilyl ether. The PMB ether is selectively removed by treatment with dichlorodicyanoquinone (DDQ) in the presence of water, and the resulting alcohol is displaced with azide with stereochemical inversion by treatment with HN3 in the presence of diethyl azodicarboxylate (DEADCAT) and triphenylphosphine. Finally, the tert-butyldiphenylsilyl group is selectively removed using buffered tetrabutylammonium fluoride, and the resulting alcohol is oxidized to the aldehyde, for example using the Dess-Martin periodinane or Swern conditions (oxalyl chloride, DMSO, triethylamine), to provide (19), fragment C wherein R2 is OTBS.
In another embodiment, fragment C wherein R2=alkoxy is prepared as illustrated in Scheme 12. 
The carbonate intermediate (17) from Scheme 8 is treated with a palladium catalyst such as tetrakis(triphenylphosphine)palladium to form the ether. The PMB protecting group is removed using DDQ, and the alcohol is converted to the azide with inversion of configuration using DEADCAT, triphenylphosphine, and HN3. The terminal aldehyde is installed as described above, yielding (20), fragment C wherein R2=alkoxy.
In another embodiment, fragment C wherein R2xe2x95x90H is prepared, that is used to prepare 20-deoxy analogs of laulimalide lactams. As illustrated in Scheme 13, these fragments are prepared using a variation of the method of Scheme 11 wherein the alcohol is first acylated, for example using acetic anhydride and a base, then reduced using a palladium catalyst and a hydride source such as triethylsilane or formic acid. 
The azide is installed as described above, followed by introduction of the terminal aldehyde to produce (21), fragment C wherein R2xe2x95x90H.
The D fragment is prepared as illustrated in Scheme 14. 
Reaction of 9-(benzoyloxy)-3,7-epoxy-non-4-enal (Paterson et al., xe2x80x9cSynthesis of the macrocyclic core of laulimalide,xe2x80x9d Org. Letts. 2001, 3: 213-216) with carbon tetrabromide and triphenylphosphine in the presence of triethylamine yields the vinyl dibromide, which is treated sequentially with n-butyllithium and methyl chloroformate to produce the acetylenic ester. The benzoyl group is removed by methanolysis, and the resulting alcohol is oxidized to the aldehyde, for example using the Dess-martin periodinane. The aldehyde is converted into the enone by reaction with dimethyl phosphonoacetone in the presence of 1,8-diazabicyclo[5.4.0]endec-7-ene (DBU) and lithium chloride to provide fragment D.
The above-described fragments C and D are used in the preparation of laulimalide lactams as illustrated in Scheme 15. 
Fragment C is coupled to fragment D using a boron-mediated aldol condensation, and the OH group of the aldol adduct is protected as its silyl ether, for example using tert-butyldimethylsilyl triflate and a mild base such as 2,6-lutidine. The ester is saponified using lithium hydroxide in water/tetrahydrofuran to provide the acid. The azide is reduced using a phosphine such as trimethylphosphine in the presence of water, and the lactam is then formed by condensation with a carbodiimide reagent such as 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI). The alkyne is partially hydrogenated to form the cis-alkene using Lindlar""s catalyst in the presence of 1-hexene. The 11-methyl group is installed by lithium dimethylcuprate addition to the enone, and the 13-ketone is converted to the 13-methylidene group using the Takai reagent, diiodomethane in the presence of zinc metal, lead iodide, and titanium tetrachloride. Removal of the silyl ether protecting groups using HFxe2x80xa2pyridine yields the 16,17-desoxylaulimalide lactam. In another embodiment of the invention, the 16,17-desoxylaulimalide lactam is converted into the laulimalide lactam by selective epoxidation using Sharpless conditions (diisopropyltartrate, titanium tetraisopropoxide, and tert-butylhydroperoxide). Use of the (+)-isomer of diisopropyltartrate provides the naturally-occurring diastereomer of the epoxide.
In another embodiment of the invention, laulimalide lactams are prepared using modifications of the method described in Ghosh et al., 2001, xe2x80x9cTotal synthesis of microtubule-stabilizing agent (xe2x88x92)-laulimalide,xe2x80x9d J. Org. Chem. 66: 8973-8982 as illustrated in Scheme 16. 
The E and F fragments are coupled using the Julia olefination sequence. The sulfone of the E fragment is deprotonated using butyllithium and added to the aldehyde of the F fragment. The resulting alcohol is acetylated, and the compound is treated with sodium amalgam to introduce the alkene. The TBS protecting group is removed by treatment with tetrabutylammonium fluoride (TBAF), and the alcohol is converted to the aceylenic ester by treatment with carbon tetrabromide, triphenylphosphine, and a base such as triethylamine to produce the vinyl dibromide, then reaction with butyllithium and methyl chloroformate to produce the acetylenic ester. The ester is saponified with LiOH in aqueous THF, and the BOC and MOM protecting groups are removed by treatment with trifluoroacetic acid. The lactam is formed by reaction of the amino acid with a carbodiimide, for example 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI). Selective reduction of the alkyne using Lindlar""s catalyst in the presence of 1-hexene provides the 16,17-desoxylaulimalide lactam. In another embodiment of the invention, the 16,17-desoxylaulimalide lactams so produced are converted into the laulimalide lactams by selective epoxidation using Sharpless conditions as described above.
(L)-homoserine lactone is elaborated into azide fragment E as illustrated in Scheme 17. 
The (L)-homoserine lactone is first N-protected, for example as the tert-butoxycarbonyl (BOC), and the lactone is opened by nucleophilic displacement with thiophenol and base. The sulfide produced is oxidized to the sulfone, for example using potassium peroxymonosulfate. The acid is then converted into the Weinreb amide through the mixed anhydride formed with methyl chloroformate and base by treatment with N,O-dimethylhydroxylamine. The R3 group is introduced by addition of the lithium acetylide to produce the acetylenic ketone. For example, use of lithium phenylacetylide results in ultimate production of the laulimalide lactam wherein R3 is phenyl. Successive reduction using L-Selectride and Red-Al provides intermediate (22), which is converted into fragment E by protection of the alcohol as the PMB ether, for example using PMB trichloroacetimidate and an acid catalyst such as pyridinium p-toluenesulfonate (PPTS).
In other embodiments of the invention, variants of fragment E wherein R2 is H or alkoxy are prepared starting from intermediate (22) using the methods described above for fragments A and C.
Typically, the inventive compound is part of a composition comprising the compound itself and a pharmaceutically acceptable carrier. Optionally, the composition may include one or more additional microtubule-stabilizing agents. Representative examples of other microtubule-stabilizing agents include but are not limited to: taxanes (e.g., paclitaxel and docetaxel), epothilone, campothecin, eleutherobin, sarcodictyins, discodermolide, and derivatives thereof. Formulations for taxanes are described by, for example, PCT publication no. WO 99/62510, which is incorporated herein by reference in its entirety.
When the composition is used to psoriasis and dermatitis, the composition optionally may contain therapeutically effective amount of one or more compounds that are used to treat psoriasis and dermatitis including but not limited to: cyclosporine; methotrexate; tamoxifen; forskolin and analogs; tar derivatives; steroids; vitamin A and its derivatives; vitamin D and its derivatives including 1-alpha-hydroxyl-cholecalciferol, 1,25-dihydrlxyl-cholecalciferol, 24, 25-dihydroxy-cholecalciferol, 1,24-dihydroxy-cholecalciferol and calcipotriol (MC 903); and beta agonists such as terbutaline.
A wide variety of carriers may be selected of either polymeric or non-polymeric origin which may be biodegradable or non-biodegradable. Representative examples of biodegradable compositions include albumin, collagen, gelatin, hyaluronic acid, starch, cellulose (methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose phthalate), casein, dextrans, polysaccharides, fibrinogen, poly(D, L-lactide), poly(D, L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters), polyesters, poly hydroxyvaleric acid), polydioxanone, poly(ethylene terephthaiate), poly(malic acid), poly(tartronic acid), polyanhydrides, polyphosphazenes, poly(amino acids) and their copolymers (see generally, Illum, L., Davids, S. S. (eds.) xe2x80x9cPolymers in Controlled Drug Deliveryxe2x80x9d Wright, Bristol, 1987; Arshady, J. Controlled Release 17: 1-22 (1991); Pitt, Int. J. Phar. 59: 173-196 (1990); Holland et al. J. Controlled Release 4: 155-180 (1986). Representative examples of nondegradable polymers include poly(ethylene-vinyl acetate) (xe2x80x9cEVAxe2x80x9d) copolymers, silicone rubber, acrylic polymers (polyacrylic acid, polymethylacrylic acid, polymethylmethacrylate, polyalkylcynoacrylate), polyethylene, polyproplene, polyamides (nylon 6,6), polyurethane, poly(ester urethanes), poly(ether urethanes), poly(ester- urea), polyethers (poly(ethylene oxide), poly(propylene oxide), Pluronics and poly(tetramethylene glycol)), silicone rubbers and vinyl polymers (polyvinylpyrrolidone. poly(vinyl alcohol), poly(vinyl acetate phthalate). Polymers may also be developed which are either anionic (e. g., alginate. carrageenin, carboxymethyl cellulose and poly(acrylic acid)), or cationic (e. g., chitosan, poly-L-lysine, polyethylenimine, and poly (allyl amine)) (see generally, Dunn et al.. J. Applied Polymer Sci. 50:353-365 (1993); Cascone et al., J. Materials Sci.: Materials in Medicine 5: 770-774 (1994); Shiraishi et al., Biol. Pharm. Bull. 16(11): 1164-1168 (1993); Thacharodi and Rao, Int""l J. Pharm. 120: 115-118 (1995); Miyazaki et al., Int""l J. Pharm. 118: 257-263 (1995)). Particularly preferred polymeric carriers include poly(ethylene-vinyl acetate), poly (D, L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomers and polymers, poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly (caprolactone), poly (valerolactone), polyanhydrides, copolymers of poly (caprolactone) or poly (lactic acid) with a polyethylene glycol (e.g., MePEG), and blends thereof.
Polymeric carriers can be fashioned in a variety of forms, with desired release characteristics and/or with specific desired properties. For example, polymeric carriers may be fashioned to release a therapeutic agent upon exposure to a specific triggering event such as pH (see e. g., Heller et al., xe2x80x9cChemically Self-Regulated Drug Delivery Systems,xe2x80x9d in Polymers in Medicine III, Elsevier Science Publishers B. V., Amsterdam, 1988, pp. 175-188; Kang et al., J Applied Polymer Sci. 48: 343-354 (1993); Dong et al., J. Controlled Release 19: 171-178 (1992); Dong and Hoffman, J. Controlled Release 15: 141-152 (1991); Kim et al., J. Controlled Release 28: 143-152 (1994); Cornejo-Bravo et al., J. Controlled Release 33:223-229 (1995); Wu and Lee, Pharm. Res 10(10): 1544-1547 (1993); Serres et al., Pharm. Res 13(2): 196-201 (1996); Peppas, xe2x80x9cFundamentals of pH- and Temperature-Sensitive Delivery Systems,xe2x80x9d in Gurny et al. (eds.), Pulsatile Drug Delivery, Wissenschaftliche Verlagsgesellschaft GmbH, Stuttgart, 1993, pp. 41-55; Doelker, xe2x80x9cCellulose Derivatives,xe2x80x9d 1993, in Peppas and Langer (eds.), Biopolymers 1, Springer-Verlag, Berlin). Representative examples of pH-sensitive polymers include poly(acrylic acid) and its derivatives (including for example, homopolymers such as poly(aminocarboxylic acid); poly(acrylic acid); poly(methyl acrylic acid), copolymers of such homopolymers, and copolymers of poly(acrylic acid) and acrylmonomers such as those discussed above. Other pH sensitive polymers include polysaccharides such as cellulose acetate phthalate; hydroxypropylmethylcellulose phthalate; hydroxypropylmethylcellulose acetate succinate; cellulose acetate trimellilate; and chitosan. Yet other pH sensitive polymers include any mixture of a pH sensitive polymer and a water soluble polymer.
Likewise, polymeric carriers can be fashioned which are temperature sensitive (see e. g., Chen et al., xe2x80x9cNovel Hydrogels of a Temperature-Sensitive Pluronic Grafted to a Bioadhesive Polyacrylic Acid Backbone for Vaginal Drug Delivery,xe2x80x9d in Proceed. Intern. Symp. Control. Rel. Bioact. Mater, 22:167-168, Controlled Release Society, Inc., 1995; Okano, xe2x80x9cMolecular Design of Stimuli-Responsive Hydrogels for Temporal Controlled Drug Delivery,xe2x80x9d in Proceed. Intern. Symp. Control. Rel. Bioact. Mater, 22: 111-112, Controlled Release Society, Inc., 1995; Johnston et al., Pharm. Res. 9(3): 425-433 (1992); Tung, Int""l J Pharm. 107: 85-90 (1994); Harsh and Gehrke, J. Controlled Release 17: 175-186 (1991); Bae et al., Pharm. Res 8(4): 531-537 (1991); Dinarvand and D""Emanuele, J. Controlled Release 36: 221-227 (1995); Yu and Grainger, xe2x80x9cNovel Thermo-sensitive Amphiphilic Gels: Poly N-isopropylacrylamide-co-sodium acrylate-co-n-N-alkylacrylamide Network Synthesis and Physicochemical Characterization,xe2x80x9d Dept. of Chemical and Biological Sci., Oregon Graduate Institute of Science and Technology, Beaverton, Oreg., pp. 820-821; Zhou and Smid, xe2x80x9cPhysical Hydrogels of Associative Star Polymers,xe2x80x9d Polymer Research Institute, Dept. of Chemistry, College of Environmental Science and Forestry, State Univ. of New York, Syracuse, N.Y., pp. 822-823; Hoffman et al., xe2x80x9cCharacterizing Pore Sizes and Water xe2x80x98Structurexe2x80x99 in Stimuli-Responsive Hydrogels,xe2x80x9d Center for Bioengineering, Univ. of Washington, Seattle, Wash., p. 828; Yu and Grainger, xe2x80x9cThermo-sensitive Swelling Behavior in Crosslinked N-isopropylacrylamide Networks: Cationic, Anionic and Ampholytic Hydrogels,xe2x80x9d Dept. of Chemical and Biological Sci.. Oregon Graduate Institute of Science and Technology, Beaverton, Oreg., pp. 829-830; Kim et al., Pharm. Res 9(3): 283-290 (1992); Bae et al., Pharm. Res 8(5): 624-628 (1991); Kono et al., J. Controlled Release 30: 69-75 (1994); Yoshida et al., J. Controlled Release 32: 97-102 (1994); Okano et al., J. Controlled Release 36: 125-133 (1995); Chun and Kim, J. Controlled Release 38: 39-47 (1996); D""Emanuele and Dinarvand. Int. J. Pharm. 118: 237-242 (1995); Katono et al., J. Controlled Release 16: 215-228 (1991); Hoffman, xe2x80x9cThermally Reversible Hydrogels Containing Biologically Active Species,xe2x80x9d in Migliaresi et al., (eds.), Polymers in Medicine III, Elsevier Science Publishers B. V., Amsterdam, 1988, pp. 161 167; Hoffman, xe2x80x9cApplications of Thermally Reversible Polymers and Hydrogels in Therapeutics and Diagnostics,xe2x80x9d in Third International Symposium on Recent Advances in Drug Delivery Systems, Salt Lake City, Utah, Feb. 24-27, 1987, pp. 297-305; Gutowska et al., J. Controlled Release 22: 95-104 (1992); Palasis and Gehrke, J. Controlled Release 18: 1-12 (1992); Paavola et al.. Pharm. Res 12(12): 1997-2002 (1995).
Representative examples of thermogelling polymers. and their gelatin temperature (LCST 0xc2x0 C.) include homopolymers such as poly(N-methyl-N-n-propylacrylamide), 19.8; poly(N-n-propylacrylamide), 21.5; poly(N-methyl-N-isopropylacrylamide), 22.3; poly(N-n-propylmethacrylamide), 28.0; poly(N-isopropylacrylamide), 30.9; poly(N-n-diethylacrylamide), 32.0; poly(N-isopropylmethacrylamide), 44.0; poly(N-cyclopropylacrylamide), 45.5; poly(N-ethylmethyacrylamide), 50.0; poly(N-methyl-N-ethylacrylamide), 56.0; poly(N-cyclopropylmethacrylamide), 59.0; poly(N-ethylacrylamide), 72.0. Moreover thermogelling polymers may be made by preparing copolymers between (among) monomers of the above, or by combining such homopolymers with other water soluble polymers such as acrylmonomers (e.g., acrylic acid and derivatives thereof such as methylacrylic acid, acrylate and derivatives thereof such as butyl methacrylate, acrylamide. and N-n-butyl acrylamide).
Other representative examples of thermogelling polymers include cellulose ether derivatives such as hydroxypropyl cellulose, 41xc2x0 C.; methyl cellulose, 55xc2x0 C.; hydroxypropylmethyl cellulose, 66xc2x0 C.; and ethylhydroxyethyl cellulose, and Pluronics such as F-127, 10-15xc2x0 C.; L-122, 19xc2x0 C.; L-92, 26xc2x0 C.; L-81, 20xc2x0 C.: and L-61, 24xc2x0 C.
A wide variety of forms may be fashioned by the polymeric carriers of the present invention, including for example, rod-shaped devices, pellets, slabs, or capsules (see e.g., Goodell et al., Am. J. Hosp. Pharm. 43: 1454-1461 (1986); Langer et al., xe2x80x9cControlled release of macromolecules from polymersxe2x80x9d. in Biomedical Polymers, Polymeric Materials and Pharmaceuticals For Biomedical Use: Goldberg, E. P., Nakagim. A. (eds.) Academic Press, pp. 113-137, 1980; Rhine et al., J. Pharm. Sci. 69: 265-270 (1980); Brown et al., J. Pharm. Sci. 72: 1181-1185 (1983); and Bawa et al., J. Controlled Release 1: 259-267 (1985)). Therapeutic agents may be linked by occlusion in the matrices of the polymer, bound by covalent linkages, or encapsulated in microcapsules. Within certain preferred embodiments of the invention, pharmaceutical compositions are provided in non-capsular formulations such as microspheres (ranging from nanometers to micrometers in size), pastes, threads of various size, films and sprays. Preferably, pharmaceutical compositions of the present invention are fashioned in a manner appropriate to the intended use. Within certain aspects of the present invention, the pharmaceutical composition should be biocompatible, and release one or more therapeutic agents over a period of several days to months. For example, xe2x80x9cquick releasexe2x80x9d or xe2x80x9cburstxe2x80x9d pharmaceutical compositions are provided that release greater than 10%, 20%, or 25% (w/v) of a therapeutic agent over a period of 7 to 10 days. Such xe2x80x9cquick releasexe2x80x9d compositions should, within certain embodiments, be capable of releasing chemotherapeutic levels (where applicable) of a desired agent. Within other embodiments, xe2x80x9clow releasexe2x80x9d pharmaceutical compositions are provided that release less than 1% (w/v) of a therapeutic agent over a period of 7 to 10 days. Further, pharmaceutical compositions of the present invention should preferably be stable for several months and capable of being produced and maintained under sterile conditions.
Within certain aspects of the present invention, pharmaceutical compositions may be fashioned in any size ranging from 50 nm to 500 xcexcm, depending upon the particular use. Alternatively, such compositions may also be readily applied as a xe2x80x9csprayxe2x80x9d, which solidifies into a film or coating. Such sprays may be prepared from microspheres of a wide array of sizes, including for example, from 0.1 xcexcm to 3 xcexcm, from 10 xcexcm to 30 xcexcm, and from 30 xcexcm to 100 xcexcm.
Pharmaceutical compositions of the present invention may also be prepared in a variety of xe2x80x9cpastexe2x80x9d or gel forms. For example, within one embodiment of the invention, pharmaceutical compositions are provided which are liquid at one temperature (e.g., temperature greater than 37xc2x0 C. such as 40xc2x0 C., 45xc2x0 C., 50xc2x0 C., 55xc2x0 C., or 60xc2x0 C.), and solid or semi-solid at another temperature (e.g., ambient body temperature, or any temperature lower than 37xc2x0 C.). Such xe2x80x9cthermopastesxe2x80x9d may be readily made given the disclosure provided herein.
Within yet other aspects of the invention, the pharmaceutical compositions of the present invention may be formed as a film, wrap or barrier. Preferably, such films are generally less than 5, 4, 3, 2, or 1 mm thick, more preferably less than 0.75 mm or 0.5 mm thick, and most preferably less than 500 xcexcm to 100 xcexcm thick. Such films are preferably flexible with a good tensile strength (e.g., greater than 50, preferably greater than 100, and more preferably greater than 150 or 200 N/cm2), good adhesive properties (i.e., readily adheres to moist or wet surfaces), and have controlled permeability.
Within further aspects of the invention, the pharmaceutical compositions may be formulated for topical application. Representative examples include: ethanol; mixtures of ethanol and glycols (e.g., ethylene glycol or propylene glycol); mixtures of ethanol and isopropyl myristate or ethanol, isopropyl myristate and water (e.g., 55:5:40); mixtures of ethanol and eineol or D-limonene (with or without water); glycols (e.g., ethylene glycol or propylene glycol) and mixtures of glycols such as propylene glycol and water, phosphatidyl glycerol, dioleoylphosphatidyl glycerol, ethyldiglycol (i.e., Transcutol(copyright)), or terpinolene; mixtures of isopropyl myristate and 1-hexyl-2-pyrrolidone, N-dodecyl-2-piperidinone or 1-hexyl-2-pyrrolidone. Other excipients may also be added to the above, including for example, acids such as oleic acid and linoleic acid, and soaps such as sodium lauryl sulfate. A preferred embodiment would include buffered saline or water, antimicrobial agents (e.g., methylparaben, propylparaben), carrier polymer(s), such as celluloses (e.g., hydroxyethylcellulose) and (a) penetration or permeation enhancer(s) (e.g., ethoxydiglycol-Transcutol(copyright), isopropyl myristate, ethylene glycol, 1 hexyl-2-pyrrolidone, D-limonene). For a more detailed description of the above, see generally, Hoelgaard et al., J. Contr. Rel. 2: 111 (1985); Liu et al., Pharm. Res. 5: 938 (1991); Roy et al., J Pharm. Sci. 83: 126 (1991); Ogiso et al., J. Pharm. Sci. 84: 482 (1995); Sasaki et al., J. Pharm. Sci. 80: 533 (1991); Okabe et al., J. Contr. Rel. 32: 243 (1994); Yokomizo et al., J. Contr. Rel. 38: 267 (1996); Yokomizo et al., J Contr. Rel. 42: 37 (1996); Mond et al., J. Contr. Rel. 33: 72 (1994); Michniak et al., J. Contr. Rel. 32: 147 (1994); Sasaki et al., J. Pharm. Sci. 80: 533 (1991); Baker and Hadgraft, Pharm. Res 12: 993 (1995); Jasti et al., AAPS Proceedings, 1996; Lee et al., AAPS Proceedings, 1996; Ritschel et al., Skin Pharmacol. 4: 235 (1991); and McDaid and Deasy, Int. J. Pharm. 133: 71 (1996).
Within certain embodiments of the invention, the pharmaceutical compositions may also comprise additional ingredients such as surfactants (e.g., 20 Pluronics such as F-127, L-122, L-92, L S 1, and L-61).
Within further aspects of the present invention, polymeric carriers are provided which are adapted to contain and release a hydrophobic compound such as a compound of the present invention. Typically, the carrier contains the hydrophobic compound in combination with a carbohydrate, protein or polypeptide. Within certain embodiments the polymeric carrier contains or comprises regions, pockets, or granules of one or more hydrophobic compounds. For example, within one embodiment of the invention, hydrophobic compounds may be incorporated within a matrix that is then incorporated within the polymeric carrier. A variety of matrices can be utilized in this regard, including for example, carbohydrates and polysaccharides such as starch, cellulose, dextran, methylcellulose, and hyaluronic acid, proteins or polypeptides such as albumin, collagen and gelatin. Within alternative embodiments, hydrophobic compounds may be contained within a hydrophobic core, and this core contained within a hydrophilic shell.
Other carriers that may likewise be utilized to contain and deliver the compounds described herein include: hydroxypropyl xcex2 cyclodextrin (Cserhati and Hollo, Int. J. Pharm. 108: 69-75 (1994)): liposomes (see e. g., Sharma et al., Cancer Res 53: 5877-5881 (1993); Sharma and Straubinger, Pharm. Res. 11(60): 889-896 (1994); WO 93/18751; U. S. Pat. No. 5,242,073), liposome/gel (WO 94/26254), nanocapsules (Bartoli et al., J. Microencapsulation 7(2): 191-197 (1990), micelles (Alkan-Onyuksel et al., Pharm. Res 11(2): 206-212 (1994)), implants (Jampel et al., Invest. Ophthalm. Vis. Science 31(11): 3076-3083(1993); Walter et al., Cancer Res. 54: 22017-2212 (1994)), nanoparticles (Violante and Lanzafame PAACR), nanoparticles-modified (U.S. Pat. No. 5,145,684), nanoparticles (surface modified) (U.S. Pat. No. 5,399,363), micelle (surfactant) (U.S. Pat. No. 5,403,858), synthetic phospholipid compounds (U.S. Pat. No. 4,534,899), gas borne dispersion (U.S. Pat. No. 5,301,464), liquid emulsions, foam, spray, gel, lotion, cream, ointment, dispersed vesicles, particles or droplets, solid- or liquid-aerosols, microemulsions {U.S. Pat. No. 5,330,756), polymeric shell (nano- and micro-capsule) (U.S. Pat. No. 5.439.686), emulsion (Tarr et al., Pharm Res. 4: 62-165, 1987), nanospheres (Hagan et al., Proc. Intern. Symp. Control Rel. Bioact. Mater. 22, 1995; Kwon et al., Pharm Res 12(2): 192-195; Kwon et al., Pharm Res. 10(7): 970-974; Yokoyama et al., J. Contr. Rel. 32: 269-277 (1994); Gref et al., Science 263: 1600-1603 (1994); Bazile et al., J Pharm. Sci. 84: 493-498 (1994)), implants (U.S. Pat. No. 4,882,168), wraps, films and inhaled formulations.
In general, methods of using the compounds of the present invention comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention. Diseases that may be treated with the compounds of the present invention are those that are characterized by cellular hyperproliferation, such as cancers, tumors, and inflammatory disorders. Illustrative examples of inflammatory disorders include, for example, atrophic gastritis, inflammatory hemolytic anemia, graft rejection, inflammatory neutropenia, bullous pemphigoid, coeliac disease, demyelinating neuropathies, dermatomyositis, inflammatory bowel disease (ulcerative colitis and Crohn""s disease), multiple sclerosis, myocarditis, myositis, nasal polyps, chronic sinusitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, surgical adhesions, stenosis or restenosis, scleritis, scleroderma, eczema (including atopic dermatitis. irritant dermatitis, allergic dermatitis), periodontal disease (i.e., periodontitis), polycystic kidney disease, and type I diabetes.
Other examples of inflammatory diseases include vasculitis (e.g., Giant cell arteritis (temporal arteritis, Takayasu""s arteritis), polyarteritis nodosa, allergic angiitis and granulomatosis (Churg-Strauss disease), polyangitis overlap syndrome, hypersensitivity vasculitis (Henoch-Schonlein purpura), serum sickness, drug-induced vasculitis, infectious vasculitis, neoplastic vasculitis, vasculitis associated with connective tissue disorders, vasculitis associated with congenital deficiencies of the complement system, Wegener""s granulomatosis, Kawasaki""s disease, vasculitis of the central nervous system, Buerger""s disease and systemic sclerosis); gastrointestinal tract diseases (e.g., pancreatitis, Crohn""s disease, ulcerative colitis, ulcerative proctitis, primary sclerosing cholangitis, benign strictures of any cause including ideopathic (e.g., strictures of bile ducts, esophagus, duodenum, small bowel or colon); respiratory tract diseases (e.g., asthma, hypersensitivity pneumonitis, asbestosis, silicosis and other forms of pneumoconiosis, chronic bronchitis and chronic obstructive airway disease); nasolacrimal duct diseases (e.g., strictures of all causes including ideopathic); and eustachean tube diseases (e.g., strictures of all causes including ideopathic).
Because the use of microtubule-stabilizing agents such as the laulimalides, taxanes, epothilones, discodermolide, eleutherobin, and the like, to treat inflammatory disorders are not as well documented as the use of microtubule-stabilizing agents to treat cancers and tumors, three representative examples of inflammatory disorders are discussed in greater detail below.
Utilizing the agents, compositions and methods provided herein, a wide variety of inflammatory skin diseases can be readily treated or prevented. For example, within one embodiment of the invention an inflammatory skin disease such as psoriasis or eczema may be treated or prevented by delivering to a site of inflammation (or a potential site of inflammation) an agent that inhibits microtubule function. Briefly, skin cells are genetically programmed to follow two possible programsxe2x80x94normal growth or wound healing. In the normal growth pattern, skin cells are created in the basal cell layer and then move up through the epidermis to the skin surface. Dead cells are shed from healthy skin at the same rate new cells are created. The turnover time (i.e., time from cell birth to death) for normal skin cells is approximately 28 days. During wound healing, accelerated growth and repair is triggered resulting in rapid turnover of skin cells (to replace and repair the wound), increased blood supply (to meet the increased metabolic needs associated with growth) and localized inflammation.
In many respects, psoriasis is similar to an exaggerated wound healing process where skin cells (called xe2x80x9ckeratinocytesxe2x80x9d) are created and pushed to the skin surface in as little as 2-4 days. Psoriasis occurs when skin cells hyperproliferate and the surface skin cannot shed the dead cells fast enough. The excess keratinocytes build up and form elevated, scaly lesions. This growth is supported by new blood vessels in the dermis (the support tissue beneath the epidermis) that are established to provide the nutrients necessary to support the hyperproliferating keratinocytes. At the same time, lymphocytes, neutrophils and macrophage invade the tissue, creating inflammation, swelling and soreness, and potentially producing growth factors that augment the rapid proliferation of the keratinocytes. All these cells (keratinocytes, vascular endothelial cells and white blood cells) produce tissue degrading enzymes or proteinases that aid in the progression of the disease and the destruction of surrounding tissue.
Utilizing the compositions of the present invention, inflammatory skin lesions may be readily treated. In particular, the microtubule-stabilizing agent is administered directly to the site of inflammation (or a potential site of inflammation), in order to treat or prevent the disease. The one or more microtubule-stabilizing stabilizing agents may be delivered as a composition along with a polymeric carrier, or in a liposome, cream or ointment formulation as discussed previously. Within preferred embodiments of the invention, the agents or compositions are delivered either topically, or by subcutaneous administration. An effective therapy for psoriasis will achieve at least one of the following: decrease the number and severity of skin lesions, decrease the frequency or duration of active disease exacerbations, increase the amount of time spent in remission (i.e., periods when the patient is symptom-free) and/or decrease the severity or duration of associated symptoms (e.g., joint pain and swelling, axial skeletal pain, bowel symptoms). Clinically the treatment will result in a reduction in the size or number of skin lesions, diminution of cutaneous symptoms (pain, burning and bleeding of the affected skin) and/or a reduction in associated symptoms (e.g., joint redness, heat, swelling, diarrhea. abdominal pain). Pathologically an microtubule-stabilizing agent will produce at least one of the following: inhibition of keratinocyte proliferation, reduction of skin inflammation (for example, by impacting on: attraction and growth factors, antigen presentation, production of reactive oxygen species and matrix metalloproteinases), and inhibition of dermal angiogenesis.
The microtubule-stabilizing agent can be administered in any manner sufficient to achieve the above end points, but preferred methods include topical and systemic administration. Patients with localized disease can be administered a topical cream, ointment or emollient applied directly to the psoriatic lesions. For example, a topical cream containing 0.001% to 10% of an inventive compound by weight is administered depending upon severity of the disease and the patient""s response to treatment. In a preferred embodiment, a topical preparation containing an inventive compound at 0.01% to 1% by weight is administered to psoriatic lesions. Alternatively, direct intracutaneous injection of an inventive compound in a suitable pharmaceutical vehicle can be used for the management of individual lesions. In patients with widespread disease or extracutaneous symptoms (e.g., psoriatic arthritis, Reiter""s syndrome, associated spondylitis, associated inflammatory bowel disease) systemic treatment can be administered. For example, intermittent treatments with an intravenous formulation can be administered at a dose of 10 to 75 mg/m2 of a compound of the present invention depending upon therapeutic response and patient tolerance. An equivalent oral preparation would also be suitable for this indication.
Other dermatological conditions that can also benefit from topical microtubule-stabilizing agents include: eczematous disease (atopic dermatitis. contact dermatitis, eczema), immunobullous disease, pre-malignant epithelial tumors, basal cell carcinoma, squamous cell carcinoma, keratocanthoma, malignant melanoma and viral warts. Topical creams, ointments, and emollients containing 0.001% to 10% inventive compound by weight can be suitable for the management of these conditions.
Microtubule-stabilizing agents may be utilized to treat or prevent chronic inflammatory neurological disorders, such as multiple sclerosis. Briefly, multiple sclerosis (xe2x80x9cMSxe2x80x9d) is a devastating demyelinating disease of the human central nervous system. Although its etiology and pathogenesis is not known, genetic, immunological and environmental factors are believed to play a role. In the course of the disease, there is a progressive demyelination in the brain of MS patients resulting in the loss of motor function. Although the exact mechanisms involved in the loss of myelin are not understood, there is an increase in astrocyte proliferation and accumulation in the areas of myelin destruction. At these sites, there is macrophage-like activity and increased protease activity which is at least partially responsible for degradation of the myelin sheath.
Microtubule-stabilizing agent of the present invention can be administered to the site of inflammation (or a potential site of inflammation), in order to treat or prevent the disease. Such agents may, within certain embodiments, be delivered as a composition along with a polymeric carrier, or in a liposome formulation as previously. Within certain embodiments of the invention, the agents or compositions may be administered orally, intravenously, or by direct administration (preferably with ultrasound, CT, fluoroscopic, MRI or endoscopic guidance) to the disease site. An effective therapy for multiple sclerosis will accomplish one or more of the following: decrease the severity of symptoms; decrease the duration of disease exacerbations; increase the frequency and duration of disease remission/symptom-free periods; prevent fixed impairment and disability; and/or prevent/attenuate chronic progression of the disease. Clinically, this would result in improvement in visual symptoms (visual loss, diplopia), gait disorders (weakness, axial instability, sensory loss, spasticity, hyperreflexia, loss of dexterity), upper extremity dysfunction (weakness, spasticity, sensory loss), bladder dysfunction (urgency, incontinence, hesitancy, incomplete emptying), depression, emotional lability, and cognitive impairment. Pathologically the treatment reduces one or more of the following, such as myelin loss, breakdown of the blood-brain barrier, perivascular infiltration of mononuclear cells, immunologic abnormalities, gliotic scar formation and astrocyte proliferation, metalloproteinase production, and impaired conduction velocity.
The microtubule-stabilizing agent can be administered in any manner sufficient to achieve the above endpoints. However, preferred methods of administration include intravenous, oral, or subcutaneous, intramuscular or intrathecal injection. The microtubule-stabilizing agent can be administered as a chronic low dose therapy to prevent disease progression, prolong disease remission or decrease symptoms in active disease. Alternatively, the therapeutic agent can be administered in higher doses as a xe2x80x9cpulsexe2x80x9d therapy to induce remission in acutely active disease. The minimum dose capable of achieving these endpoints can be used and can vary according to patient, severity of disease, formulation of the administered agent, and route of administration. For example, preferred embodiments would include 10 to 75 mg/m2 of an inventive compound once every 1 to 4 weeks, 10 to 75 mg/m2 daily, as tolerated, or 10 to 175 mg/m2 once weekly, as tolerated or until symptoms subside.
Inflammatory arthritis is a serious health problem in developed countries, particularly given the increasing number of aged individuals. For example, one form of inflammatory arthritis, rheumatoid arthritis (xe2x80x9cRAxe2x80x9d) is a multisystem chronic, relapsing, inflammatory disease of unknown cause. Although many organs can be affected, RA is basically a severe form of chronic synovitis that sometimes leads to destruction and ankyiosis of affected joints (Robbins Pathological Basis of Disease, by R. S. Cotran, V. Kumar, and S. L. Robbins, W. B. Saunders Co., 1989). Pathologically, the disease is characterized by a marked thickening of the synovial membrane which forms villous projections that extend into the joint space, multilayering of the synoviocyte lining (synoviocyte proliferation), infiltration of the synovial membrane with white blood cells (macrophages, lymphocytes, plasma cells, and lymphoid follicles; called an xe2x80x9cinflammatory synovitisxe2x80x9d), and deposition of fibrin with cellular necrosis within the synovium. The tissue formed as a result of this process is called pannus and, eventually the pannus grows to fill the joint space. The pannus develops an extensive network of new blood vessels through the process of angiogenesis that is essential to the evolution of the synovitis. The release of digestive enzymes (matrix metalloproteinases such as collagenase, stromelysin, and the like) and other mediators of the inflammatory process (e.g., hydrogen peroxide, superoxides, lysosomal enzymes, and products of arachadonic acid metabolism) from the cells of the pannus tissue leads to the progressive destruction of the cartilage tissue. The pannus invades the articular cartilage leading to erosions and fragmentation of the cartilage tissue. Eventually there is erosion of the subchondral bone with fibrous ankylosis and ultimately bony ankylosis, of the involved joint. It is generally believed, but not conclusively proven, that RA is an autoimmune disease, and that many different arthrogenic stimuli activate the immune response in the immunogenetically susceptible host. Both exogenous infectious agents (Ebstein-Barr virus, rubella virus, cytomegalovirus, herpes virus, human T-cell lymphotropic virus, Mycoplasma, and others) and endogenous proteins (collagen, proteoglycans, altered immunoglobulins) have been implicated as the causative agent that triggers an inappropriate host immune response. Regardless of the inciting agent, autoimmunity plays a role in the progression of the disease. In particular, the relevant antigen is ingested by antigen-presenting cells (macrophages or dendritic cells in the synovial membrane), processed, and presented to T lymphocytes. The T cells initiate a cellular immune response and stimulate the proliferation and differentiation of B lymphocytes into plasma cells. The end result is the production of an excessive inappropriate immune response directed against the host tissues (e.g., antibodies directed against type II collagen, antibodies directed against the Fc portion of autologous IgG (called xe2x80x9cRheumatoid Factorxe2x80x9d)). This further amplifies the immune response and hastens the destruction of the cartilage tissue. Once this cascade is initiated numerous mediators of cartilage destruction are responsible for the progression of rheumatoid arthritis.
Thus, within one aspect of the present invention, methods are provided for treating or preventing inflammatory arthritis (e.g., rheumatoid arthritis) comprising the step of administering to a patient a therapeutically effective amount of an microtubule-stabilizing agent. Inflammatory arthritis includes a variety of conditions including, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis (scleroderma), mixed connective tissue disease, Sjogren""s syndrome, ankylosing spondylitis, Behcet""s syndrome, sarcoidosis, and osteoarthritisxe2x80x94all of which feature inflamed, painful joints as a prominent symptom. Within a preferred embodiment of the invention, microtubule-stabilizing agents may be administered directly to a joint by intra-articular injection, as a surgical paste or administered by another route, e.g., systemically or orally. Such agents may, within certain embodiments, be delivered as a composition along with a polymeric carrier, or in a liposome formulation as discussed previously.
An effective microtubule-stabilizing therapy for inflammatory arthritis will accomplish one or more of the following: (i) decrease the severity of symptoms (pain, swelling and tenderness of affected joints; morning stiffness. weakness, fatigue. anorexia, weight loss); (ii) decrease the severity of clinical signs of the disease (thickening of the joint capsule, synovial hypertrophy, joint effusion, soft tissue contractures, decreased range of motion, ankylosis and fixed joint deformity); (iii) decrease the extra-articular manifestations of the disease (rheumatic nodules, vasculitis, pulmonary nodules, interstitial fibrosis, pericarditis, episcleritis, iritis, Felty""s syndrome, osteoporosis); (iv) increase the frequency and duration of disease remission/symptom-free periods; (v) prevent fixed impairment and disability; and/or (vi) prevent/attenuate chronic progression of the disease. Pathologically, an effective microtubule-stabilizing therapy for inflammatory arthritis will produce at least one of the following: (i) decrease the inflammatory response, (ii) disrupt the activity of inflammatory cytokines (such as IL-I, TNFa, FGF, VEGF), (iii) inhibit synoviocyte proliferation, (iv) block matrix metalloproteinase activity, and/or (v) inhibit angiogenesis. An microtubule-stabilizing agent will be administered systemically (orally, intravenously, or by intramuscular or subcutaneous injection) in the minimum dose to achieve the above mentioned results. For patients with only a small number of joints affected, or with disease more prominent in a limited number ofjoints, the microtubule-stabilizing agent can be directly injected (intra-articular injection) into the affected joints. The microtubule-stabilizing agent can be administered in any manner sufficient to achieve the above endpoints. However, preferred methods of administration include intravenous, oral, or subcutaneous, intramuscular or intra-articular injection. The microtubule-stabilizing agent can be administered as a chronic low dose therapy to prevent disease progression, prolong disease remission, or decrease symptoms in active disease.
Alternatively, the therapeutic agent can be administered in higher doses as a xe2x80x9cpulsexe2x80x9d therapy to induce remission in acutely active disease. The minimum dose capable of achieving these endpoints can be used and can vary according to patient, severity of disease, formulation of the administered agent, and route of administration. For example, preferred embodiments would include 10 to 75 mg/m2 of an inventive compound once every 1 to 4 weeks, 10 to 75 mg/m2 daily, as tolerated, or 10 to 175 mg/m2 once weekly, as tolerated or until symptoms subside.
A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the present invention and shall not be construed as being a limitation on the scope of the invention or claims.